While combination antiretroviral therapy (cART) is effective at suppressing HIV, it fails to eradicate latent provirus or restore virus-specific T-cel responses that can eliminate virus-expressing cells after induction with a latency reversing agent or treatment interruption. One potential way is to create designer HIV-specific T- cells for use by adoptive T-cell therapy, which has shown promise for treating other viral infections and cancer in clinical trials. In this application, we proposed to develop a chimeric antigen receptor (CAR) specific for the HIV-1 envelope protein (Anti-HIV-1 Env-CAR), which when expressed on modified T-cells, is capable of protecting the modified T-cells from infection and triggering T-cel responses required for the clearance of infected cells. Two findings serve as the basis for the anti-HIV-1 Env-CAR. First, our past studies have demonstrated that some single chain variable fragments (scFvs) of anti-HIV Env antibodies are potent inhibitors of HIV-1 when expressed on the surface of target cells. Second, the co-investigator and others have demonstrated the potential for adoptive T-cell therapy for treating viral infections and cancer, and the promise for CARs to improve persistence and anti-tumor activity of engineered T-cells. They have also demonstrate the capacity to expand broadly-specific polyclonal T-cells that can clear HIV-infected cells. We hypothesize that combining a neutralizing anti-HIV Env scFv with a second generation CD28?-CAR having activation and costimulatory properties will result in a bifunctional CAR (anti-HIV Env-CD28?) molecule that promotes proliferation and anti-HIV T-cell responses in modified T-cells while protecting them from HIV infection and cell death. This CAR could be used to recognized and eliminate latently infected cells after induction of viral gene expression with a latency reversing agent. The hypothesis will be addressed in three specific aims. Aim1 will examine whether ex vivo expanded T-cells modified with the anti-HIV Env-CD28? CAR will direct Env-specific T-cell responses and killing of infected target cells. Aim2 wil examine whether the anti-HIV Env-CD28? protects modified T-cells from infection by diverse HIV-1 variants. Aim3 will determine whether anti-Env- CD28? CAR modified T-cells enhance HIV-1 infected cell clearance and decrease viral loads in a humanized mouse model. Successful completion of this project will advance the potential for using CAR modified T-cells for targeting and eliminating latent HIV reservoirs after reactivation, a primary goal of the TaPHIR R21/R33 program.